Setting of a procedure for experimental incubation of Pacific oyster (Crassostrea gigas) embryos

Standardized experimental protocols designed to study Pacific oyster (Crassostrea gigas) gamete quality have not been previously published. Gamete sampling variations and confounding effect of interacting factors result in large variations between replicates, decreasing the effects of the studied factors. This work aims at defining a standardized procedure for incubation of oyster embryos designed for experimental purposes. In a first phase, four experiments were developed to improve embryo sampling and handling. They showed that a minimum of 50 embryos must be counted to decrease the variation between counting replicates. For reliable results, sampling must be carried out from 0 to 7 min after a careful agitation of seawater containing embryos (salinity: 35.6[per thousand]). Compared to control and a 30 cm one, the D-larval yield of embryos submitted to a 1 m fall was significantly reduced, showing the limited effect of mechanical disturbance on Pacific oyster oocytes survival. Then, maintaining 2.2 million embryos in 250 ml seawater for a two hours period resulted in a decrease of D-larval yield which could not be explained by a decrease of O2 content and pH of seawater. The second phase of this work included a set of five experiments, defining experimental incubation conditions. A higher larval yield was observed using 1.8 l beakers and 150 l tanks compared to 0.3 ml microtiter plates and 1 l fish egg incubators. Higher larval yields were recorded when embryo density ranged from 5 to 100 ml- 1, compared to values between 500 and 2000 ml- 1. Compared to controls (no antibiotic or no presence of light), no changes of D-larval yield were observed by adding 20 ppm chloramphenicol or by maintaining embryos in total darkness. Then, a significant decrease of pH and O2 content was observed during the incubation period. However, these changes could not be considered as limiting for Pacific oyster embryo development. In conclusion, experimental incubation conditions have been defined in this study: 30 embryos ml- 1 incubated in 1.8 l beakers without antibiotic and regardless of light intensity, when not higher than 500 lux. The mean coefficients of variation observed between tank replicates ranged from 13.1 to 16.5%. The standardized incubation procedure described in this work will help to study quality variations of Pacific oyster gametes.